The use of lipases or esterases to stereoselectively hydrolyze esters has received considerable attention over the past several years. See Cambou B. and A. M. Klibanov, Lipase-Catalyzed Production of Optically Active Acids via Asymmetric Hydrolysis of Esters/Effect of the Alcohol Moiety, Appl Biochem & Biotech 9, 225-260 (1984); Dahod, S. K. and P. Siuta-Mangano, Resolution of Racemic Mixture of Aliphatic Acid Esters, Stauffer Chemical Company, European Patent Application 86104201.8 (03-26-1986) (hereinafter Dahod EPA); Dahod, S. K. and P. Siuta-Mangano, Carbon Tetrachloride-Promoted Stereo Selective Hydrolysis of Methyl-2-Chloropropionate by Lipase, Biotech. & Bioeng. 30, 995-999 (1987) (hereinafter Dahod Biotech); and Walts, A. E., Fox, E. M., and C. B. Jackson, R-Glycidyl Butyrate: Evolution of A Laboratory Procedure to an Industrial Process, Biotech USA 1987, Online International, London. This is due to a desire to produce agricultural or pharmaceutical intermediates with a particular stereo-chemistry where the undesired enantiomer possesses little or none of the activity, but may be responsible for unwanted toxicity. See Calton, G. J., Use of Microorganisms and Enzymes in the Synthesis and Production of Optically Active Agricultural Chemicals, ACS Symposium Series 334 Biotechnology in Agricultural Chemistry, Amer. Chem. Soc., Washington D.C., 1987. A number of propionic acid derivatives which are used in the agricultural and pharmaceutical fields fall into this category. See Dahod EPA, Dahod Biotech; and Calton, and Caldwell, J., Hutt, A. J., and Fournel-Gigleux, S., Metabolic Chiral Inversion and Dispositional Enantioselectivity of the 2-Arylpropionic Acids and Their Biological Consequences, Biochemical Pharmacology 37, 105-114 (1988).
Although a large number of lipases have been evaluated for the resolution of racemic alcohols and/or carboxylic acids, they have not generally met with great success due to enantiospecificities below 95%. It is generally accepted that enantioselectivities above 95% are required for an industrially significant process. See Akiyama, A., Bednarski, M., Kim, M.-J., Simon, E. S., Waldmann, H. and Whitesides, G. M., Enzymes in Organic Synthesis, Chemtech 1988, 627-634 (hereinafter Whitesides). Only a few of the lipases reported in the literature exhibit a high degree of stereospecificity. See Dahod EPA; Dahod Biotech; Walts, Nissan Chem Ind Japanese Pat Appl 8 6-20283/31; Stokes, T. M. and A. C. Oehlschlager, Enzyme Reactions in Apolar Solvents: The Resolution of (.+-.)-Sulcatol With Porcine Pancreatic Lipase, Tetra. Lett. 28, #19, 2091-2094 (1987); Kodera, Y., Takahashi, K., Nishimura, H., Matsushima, A., Saito, Y., and Y. Inada, Ester Synthesis from -substituted Carboxylic Acid Catalyzed by Polyethylene Glycol-Modified Lipase From Candida rugosa in Benzene, Biotech. Lett. 8, 881-884 (1986); and Klibanov, A. M. and Kirchner, G. Enzymatic Production of Optical Isomers of 2-Halopropionic Acids, U.S. Pat. No. 4,601,987, Jul. 22, 1986. Lipases have also been extensively examined for the production of optically active esters from mixtures of acids and/or alcohols and have exhibited good enantioselectivity for esterification with, long chain alcohols. See Klibanov, A. M. and Kirchner, G., Enzymatic Production of Optical Isomers of 2-Halopropionic Acids, U.S. Pat. No. 4,601,987, Jul. 22, 1986; and Cambou B. and Klibanov, A. M., Preparative Production of Optically Active Esters and Alcohols Using Esterase-Catalyzed Stereospecific Transesterification in Organic Media, J. Am. Chem. Soc. 106, 2687-2692 (1984).
The lipase from Candida rugosa (formerly known as Candida cylindracea) has been used extensively and has been shown to exhibit limited stereospecificity in the hydrolysis of some esters. This enzyme is available commercially from a large number of sources, including Sigma, Meito Sangyo, Biocatalysts, Enzyme Development Corp and Amano. Its production is based on extraction from fermentation broths and cells of C. rugosa ATCC #14830. Prior work has shown that the lipase from Candida rugosa (Sigma) did not give stereospecific hydrolysis of methyl esters of 2-chloropropionic acid but did give a high enantiomeric excess in the hydrolysis of the octyl ester of 2-chloropropionic acid. See Klibanov, A. M. and Kirchner, G., Enzymatic Production of Optical Isomers of 2-Halopropionic Acids, U.S. Pat. No. 4,601,987, Jul. 22, 1986. Additionally, it has been shown that the synthesis of the octyl ester was absolutely stereospecific. See Cambou B. and Klibanov: A. M., Preparative Production of Optically Active Esters and Alcohols Using Esterase-Catalyzed Stereospecific Transesterification in Organic Media, J. Am. Chem. Soc. 106, 2687-2692 (1984).
It has also been found that in a two phase aqueous/organic mixture at low temperatures (4.degree. C.), one can obtain high optical purity (90% ee) after 30% hydrolysis. Interestingly, heavily chlorinated solvents were superior for this hydrolysis and CCl.sub.4 activated the enzyme in the presence of substrate, whereas in the absence of substrate, CCl.sub.4 rapidly deactivated the enzyme. See Dahod and SiutaMangano. It has also been shown that the optical purity obtained in the reaction was a function of the extent of hydrolysis, in accordance with earlier predictions and that optical purity decreased rapidly as the reaction neared complete hydrolysis of one of the enantiomers.
Sih and co-workers, (Sih, C. J., Gu, Q-M, Fulling, G., Wu, S.-H and Reddy, D. R. J. Indus. Micro Suppl 3, Develop Ind Micro., 1988, 29, 221-229; Gu, Q.-M., Chen, C.-S. and Sih, C. J. Tet Lett 1986, 27, 1763-1766) have found excellent resolution of the (S) enantiomer of 2-arylpropionic acids, depending on the nature of the aryl substituent. For 2-(6-methoxy-2-naphthyl)propionic acid (naproxen) and p-isobutylhydratropic acid (ibuprofen), the enantiospecificity of Candida rugosa lipase was over 98% ee. However, for 2-(3-benzoylphenyl)propionic acid (ketoprofen), the ee was only 51%.
Workers at Nissan Chemical Industries (Nissan Chem Ind Japanese Patent JP-255917 1986) attempted to take advantage of the stereoselective hydrolysis by a preparation of Candida rugosa lipase for the production of an aryloxypropionic acid, (R)-(+)-2-(4-hydroxyphenoxy)propionic acid (R-HPPA); however, they only obtained an 87% ee at 21% hydrolysis in aqueous solution.
The lipase of Candida rugosa has been referred to as if it were a single enzyme regardless of its source although recently a separation of the crude enzyme has been published. See Abramowicz, D. A. and Keese, C. R., Enzymatic Transesterifications of Carbonate in Water-Restricted Environments, Biotech. Bioeng. 33, 149-156 (1989). However, it is well known in the literature that many enzymes contain a number of different isozymes with different specifications. Isozymes from pig liver esterase have been isolated. See Farb, D. H., Multiple Forms of Microsomal Porcine Liver Esterase: I. Isolation and Properties, Estimation of Secondary Structure. II. pH Dependence of Rate Contacting Groups, Diss Ab. Int B 38, 5900-5901 (1978); Farb, D. and Jencks, W. P., Dependence on pH of the Activity of Pig Liver Esterase. Arch. Biophy. 203, 227-35 (1980); and Guanti, G., Banfi, L., Narisano, E., Riva, R. and Thea, S., Enzymes in Asymmetric Synthesis: Effect of Reaction Media on the PLE Catalyzed Hydrolysis of Diesters, Tet. Let. 27, 4639-4642 (1986). Depending on the substrate, each of these isozymes may react differently to changes in the reaction conditions, and may give differing stereospecificity. However, no indication of isozyme existence in C. rugosa lipase exists, despite the fact that the enzyme has been extensively investigated even with isoelectric focusing. See Gerlach, D., Schneider, S., Gollner, T., Kim, K. S., and Schreier, P., Screening of Lipases for Enantiomer Resolution of Secondary Alcohols by Esterification in Organic Medium, Bioflavour '87, Proc. Int. Conf., 1988 Walter de Gruyter & Co., Berlin.
Notwithstanding the foregoing prior efforts, there is still considerable room for providing improved procedures for stereoselectively hydrolyzing or transesterifying esters or for esterifying alcohols or esters by enzymatic means. For example, there is a continuing interest in the enzymatic hydrolysis of (R,S)-(+)-methyl-2-(4-hydroxyphenoxy)-propionate (HPPA-Me) to produce (R)-HPPA in greater than 95% ee. Racemic HPPA is a valuable intermediate in the production of certain herbicides. Over 95% of he herbicidal activity of quizalofop ethyl has been found to lie in the (R)-isomer which is conveniently synthesized from (R)-HPPA. It, therefore, is of considerable significance to be able to resolve the racemic ester of HPPA in an economic fashion to provide this intermediate on an industrial scale. A purpose of the invention is to provide such improvements in enzymatic hydrolysis, esterification and transesterification procedures. Other objects will also be hereinafter evident.